Human embryonic stem cells (hESCs)1 and human induced pluripotent stem cells (hiPSCs)2,3 are self-renewing pluripotent cells that are able to differentiate into many cell types in the body. They hold great promises e.g. for cell therapy, drug research and tissue engineering. Further, it is envisioned in the future human induced pluripotent stem cells, multipotent cells and other undifferentiated cells will be proliferated and directed to differentiate into specific lineages so as to develop differentiated cells or tissues which can be transplanted into human bodies for therapeutic purposes. Human pluripotent stem cells and the differentiated cells that may be derived from them are also powerful scientific tools for studying human cellular and developmental systems.
In order to expand hESCs and hiPSCs and prevent their spontaneous differentiation, some in vitro culture systems have been developed. Conventionally the cells were cultured in these systems on feeder cells1 and later Matrigel coating4 was introduced to replace feeder cells in combination with the use of conditioned medium or chemically defined medium, for example, mTeSR1 medium5. Matrigel, however, includes poorly defined matrix components and reproduction of optimised cell cultures is difficult because of batch-to-batch variation of the material. More recently, several studies using vitronectin (VN)6, laminin-511 (LM-511)7, LM-521, synthetic peptide-acrylate surface8 or synthetic polymer coating9 have made progress in developing chemically defined in vitro culture systems for propagation of hESCs and hiPSCs at undifferentiated state. However, all these culture systems are using two-dimensional (2D) surfaces, which do not mimic the in vivo environment of stem cells, called stem cell niche. In addition, cells cultured on 2D surfaces are not easily scalable to larger quantities required for e.g. therapy and research.
Adult stem cells, or somatic stem cells, are undifferentiated cells found throughout the body after differentiation. They are responsible for e.g. organ regeneration and capable of dividing in pluripotent or multipotent state and differentiating into differentiated cell lineages.
Human mesenchymal stem cells (hMSC) display a very high degree of plasticity and are found in virtually all organs with the highest density in bone marrow. HMSCs serve as renewable source for mesenchymal cells and have pluripotent ability of differentiating into several cell lineages, including osteoblasts, chondrocytes, adipocytes, skeletal and cardiac myocytes, endothelial cells, and neurons in vitro upon appropriate stimulation, and in vivo after transplantation.
The stem cell niche is a well-defined complex 3D microenvironment and it regulates stem cell fate by spatially presenting biochemical and physical signals. The cells under physiological conditions not only “cross-talk” between each other but also interact with their cellular microenvironment and the extra-cellular matrix (ECM). The ECM provides structural support to the cells and also contributes to signalling and directing cell fate. Mostly, the ECM is composed of glycosaminoglycans and fibrous proteins such as collagen, elastin, laminin and fibronectin self-assembled into nanofibrillar network.
In 3D cell culturing, a suitable culturing matrix should be able to mimic components of native ECM to provide a scaffold having similar properties with the native ECM, such as structural support for cells and a network of interconnected pores for efficient cell migration and transfer of nutrients to the cells.
Hydrogels, both of synthetic and natural origin, have recently emerged as suitable scaffolds for 3D cell culture. The network of interconnected pores in hydrogels allows retention of a large amount of biological fluid facilitating transport of oxygen, nutrients and waste. Furthermore, most hydrogels can be formed under mild cytocompatible conditions and their biological properties can be modulated by surface chemistry.
Engineered hydrogels with modified mechanical, chemical and biological properties have the potential to mimic the ECM and thus establish their utility in 3D cell culture. Commercial products for 3D cell culturing are for example cell culture matrices PuraMatrix™ (3DM Inc.) and Matrigel (BD Biosciences). PuraMatrix™ is a hydrogel of self-assembled peptide nanofibers which resembles the structure of natural fibrillar collagen in ECM with fiber diameter 5-10 nm. It has also high water content, typically 99.5%. U.S. Pat. No. 7,449,180 and WO 2004/007683 disclose peptide hydrogels. Matrigel is gelatinous protein mixture secreted by mouse tumor cells. The mixture resembles the complex extracellular environment found in many tissues and is used by cell biologists as a substrate for cell culture. MaxGel® ECM Matrix (Sigma-Aldrich), which includes a mixture of human ECM components, forms a gel in ambient temperature. Typically, in these systems the pluripotent cells are separated from the cell culture matrix by protease treatment which breaks extracellular protein network used by the cells to attach themselves to the cell culture matrix and to neighbouring cells.
Bacterial cellulose (BC) has been used in wound healing membranes and as a scaffold in cell culture. The limitation in the use of bacterial cellulose in stem cell culture is the inherent structure of the fermented material: Upon cultivation, BC is formed as very tight membranes in air-water interphase in the fermenter. The formed membranes are too tight for 3D cell culturing and various modifications. If used as cell culture matrix, the porosity of the BC matrix has to be increased for adequate cell penetration and formation of cell clusters.
U.S. Pat. No. 5,254,471 discloses a carrier for cell culture comprising ultra fine fibers. WO 2009/126980 discloses cellulose-based hydrogels whose framework substance consists essentially of or entirely of cellulose and are formed by regeneration from organic solvents. EP1970436B1 discloses carrier material for undifferentiated cell cultures. Present 2D and 3D cell culture systems for pluripotent cell cultures, such as stem cells, rely on animal based matrices. Animal based compounds in cell culture environment generate a risk of immunoreactions and different types of toxicity issues in cell culture and downstream applications. Further, harvesting cells from cell culture matrices composed of proteinaceous material requires treating the cell culture with protein degrading enzyme such as protease, which is also hydrolyses extracellular structures of the cultured cells.